Liver-specific adenovirus expression vector

ABSTRACT

The invention concerns an adenovirus vector for liver specific gene  thera fields of application in medicine are the treatment of gene defects and tumour diseases of the liver and molecular biology. The vector according to the invention is marked by the fact that a therapeutic gene is coupled with a liver-specific promoter consisting of enhancer elements of the hepatitis B virus and an enhancerless minimum promoter and is optionally surrounded by SAR elements wherein the promoter is inserted into the adenovirus genome.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a United States national stage of PCT/DE96/01253,filed Jul. 8, 1996.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A "MICROFICHE APPENDIX"

Not applicable.

BACKGROUND OF THE INVENTION

The invention relates to an adenovirus vector for liver-specific genetherapy. Fields of application include medicine, the treatment of genedefects and tumour diseases of the liver, and molecular biology.

Numerous methods and vectors for gene therapy have been developed inrecent years (survey in Mulligan/1993/Science 260, 920). Vectors derivedfrom viruses are compared with those from non-viral transfer methodswhere the therapeutic gene is embedded in protein or lipid coats. Theparticles derived from the non-viral methods can preferentially bind tospecific receptors once the particles are coupled to ligands for thesereceptors. This specificity works as an advantage to the genetherapeutic system in vivo. However, the therapeutic gene reaches onlythe tissue where its activity is desired.

Yet, viral vectors, notably the virus groups retrovirus and adenovirushave shown a higher efficiency in vivo. Both viruses allow the transferof genes in liver cells. The retroviral infection leads to a stableintegration of the genetic material into the cellular genome. Thisprocess depends on the proliferation of cells--a rare event for livercells in vivo. Hepatocytes in culture are infected with retrovirusessufficiently. In the liver, however, either a partial liver resection isnecessary to stimulate cell division (Ferry et al./1991/Proc. Natl.Acad. Sci. USA 88, 8377) or the application of methods which result inan acute decrease in the number of hepatocytes (Lieber et al. Proc.Natl. Acad. Sci. USA in press) is required to produce the same results.

Adenoviruses are by far more superior and more efficient to all othertypes of vectors. Even if administered intravenously, the virus mayreach nearly 100 percent of the hepatocytes. Adenoviruses are availableas episomes in the cell.

In contrast to retrovirus vectors, adenoviral vectors contain thebiggest part of the viral genome. Originally, only the E1 region wastransferred to the helper cell (HEK293) and used for the multiplicationof viruses, thus preventing the virus from being replicated in thetarget tissue. As the adenovirus coat may receive up to 105 percent ofthe size of a genome (40 kb) the deletion of E1 was also essential forthe insertion of new genes. To increase the capacity to a maximum of 8.3kb, parts of the E3 region were additionally deleted (Bett etal./1994/Proc. Natl. Acad. Sci. USA 91, 8802). Although the mostimportant transactivators of adenoviral genes--products of the E1region--are lacking, other viral genes are expressed in addition to thetherapeutic gene. The exposition of the respective proteins on the cellsurface results in an activation of CD8 positive T-cells and anelimination of the infected cells. By eliminating furthertransactivators, e.g. E2A, it was functionally possible to reduce thiseffect farther (Yang et al./1994/Nature Genetics 7, 362).

An essential drawback of the existing adenovirus vectors is the lack oftissue specificity. Adenovirus receptors exist in a multitude of celltypes, thereby explaining the lack of enthusiasm for methods calling forthe additional coupling of the virus with ligands of specific receptors.

Liver specificity may also be achieved by using liver-specific promotersapart from the reception mode. Various cellular promoters (albumin andalphal antitrypsin promoters) active in hepatocytes were examined forgene therapy in retroviruses (Rettinger et al./1994/Proc. Natl. Acad.Sci. USA 91, 1460). Their size, however, makes them unsuitable inadenovirus vectors. Furthermore, the strong viral promoters (CMV and RSVpromoters), frequently applied in the adenoviral context, areubiquitously active and eliminated in the liver after a short time.

BRIEF SUMMARY OF THE INVENTION

This invention is aimed at constructing a vector that combines theadvantages of an adenovirus vector with the property of liver-specificexpression of therapeutic genes.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 Expression of the luciferase reporter gene controlled by variouspromoters in hepatocyte cell lines and cell lines of a different origin,measured as activity of the luciferase encyme.

The activity is given as percent (%) of the activity achieved with theCMV promoter. The values given are averages from 4 independenttransfection experiments.

FIG. 2 Construction of adenoviruses expressing the gene of the human LDLreceptor under the control of promoters derived from CMV or HBV.

CMV, CMV immediate early promoter; CP, HBV core promoter; EI, EBCenhancer I; EII, HBV enhancer II; mCMV, minimum CMV promoter; polyA;polyadenylation signal of the bovine growth hormone

FIG. 3 Expression of LDL receptors in cell lines after an adenoviralgene transfer

FIG. 4 (Parts A-C Expression of the human receptor gene in vivo

Samples of the respective tissue were powdered and used in equal amountsfor isolating RNA and genomic DNA. Ten (10) μg of the total RNA werealways used in the RNA protection assay. Here, an anti-sense RNA of theLDL receptor organ or the GAPDH gene synthesised in vitro by means of T7RNA polymerase using ³² p-marked nucleotides serves as a sample. It isprotected against RNAse digestion over a length of 304 bp (LDLR) or 316bp (GAPDH) of the respective cellular transcript.

Genomic DNA was analysed using Southern Blotting for analysing theefficiency of infection. Ten (10) μg of genomic DNA were digested withNco1 and separated in agarose gel. In this connection, a 1584 bp longfragment will be released in the 5' region of the adenovirus which willbe detected by hybridisation by means of a ³² marked DNA probe (equalfragment).

FIG. 5 (Parts A-D) Expression of the human LDL receptor gene in vivo bymeans ofRT-PCR

a) Schematic representation of the principle of the competitive RT-PCR.When amplifying a LDLR-RNA transcribed in reverse order from the tissuea 450 bp long fragment is obtained. A 267 bp long fragment is amplifiedfrom the shortened LDLR-RNA synthesised in vitro after reversetranscription.

b,c,d) Equal quantities of RNA from the respective tissue were mixedwith increasing quantities of the shortened transcript, transcribed inreverse order and subjected to a PCR by ³² marked nucleotides over 30cycles. After having been separated in agarose gel on the phosphoimmager(Fuji) the PCR products were analysed.

The triangles show the mixing ratio where PCR products are available inequivalent quantities.

DETAILED DESCRIPTION OF THE INVENTION

An essential component of the invention is a short, highly active andliver-specific promoter which is coupled to the therapeutic gene. Itconsists of enhancer elements of the hepatitis B virus and anenhancerless minimum promoter. Moreover, it is necessary to surround thepromoter with SAR elements (scaffold attached regions), in order toprotect the inserted gene against the effects of adenoviral enhancers.The SAR region of the human interferon-β-gene is preferentially used assuch an element.

Enhancer II of the hepatitis B virus (subtype ayw) is preferentiallyused as an enhancer element. It is detected by positions 1628-1807 onthe viral gene.

A part of the former promoter of the cytomegalovirus is used as theenhancerless minimum promoter. It is located at positions -55 to +69with respect to the start of transcription. Essential elements of theminimum promoter are the TATA box, the "cap place" and the5'-non-translated region. The minimum promoter has a low basicexpression and may be activated by enhancer II of the hepatitis B virus.

According to the invention, the preferential place for the insertion ofthe expression unit consisting of the enhancer, promoter and gene isregion E1 of the adenogenome. Notably, subtypes 2 and 5 of this virusare used as adenoviruses.

The cDNA of the human LDL receptor is used as the therapeutic gene. Thereceptor is normally expressed at a high rate in hepatocytes. Inpatients with the disease family hypercholesterolaemia this gene ismutated. The therapy requires a high expression of the receptor as wellas a lasting effect in these cells.

The cDNAs of other genes may replace or substitute the cDNA of the LDLreceptor if these genes are defective in the disease to be cured, andthey are mainly active in the liver.

The advantages of the vector in conformity with the invention are thehigh efficiency of the infection combined with a high specificity ofexpression in liver cells. In addition, the promoter remains active inthe liver for a long time if it is used in adenovirus vectors with areduced immunogenity. Thus, it may be used in a more efficient therapyof congenital gene defects of the liver.

Hereinafter the invention is explained in further detail by thefollowing illustrative execution thereof

Example of Execution

In vivo strategies of the liver gene therapy require hepatocyte-specificvectors.

Owing to their high titre and stability in blood adenoviruses areespecially suited for an in vivo gene transfer. The natural target cellsfor adenoviruses are cells of epithelial tissue. There is no specialtropism for hepatocytes. Thus, special ways of application ormodifications of the proper virus are performed for organ specificity.

The modification of virus proteins for the selective infection ofhepatocytes does not appear to be promising due to the complexity of thevirus coat and the virus-receptor interaction. Yet, alternatively theexpression of the transferred therapeutic gene may be restricted tohepatocytes by using solely liver-specific promoters.

Cellular promoters with a known liver specificity (albumin promoters,alphal antitrypsin promoters) are not suitable for use as adenoviralvectors due to their size or are not considered for the strict metaboliccontrol taking place (PEPCK promoter).

The hepatitis B virus has a genome of 3.2 kb, and its genes arecontrolled by 4 various promoters (preS1, S, core and X promoters). Inaddition, they are activated, to a differing degree, by 2 enhancers(enhancers I and II). All of these controlling elements comprise only afew hundred base pairs.

These promoters/enhancers were obtained from the cloned genome of thehepatitis B virus (subtype ayw) through PCR, and subsequently cloned infront of a promoterless luciferase gene. The promoters were examined fortheir liver specificity in a transient liver cell line test(determination of the activity of luciferase) in the liver cell linesHuH7 and HepG2 and HepSV40 and the non-liver cell lines NIH3T3, HeLa andCV1. In addition, the promoter strength was compared with that of theCMV promoter. Transfection was effected by means of the Ca(PO₄)₂precipitation technique and was standardized by coprecipitation of aβ-galactosidase gene controlled by a RSV promoter and measurement of theactivity of β-galactosidase (FIG. 1).

Based upon these expression studies, the chosen promoter/enhancer II(pCPluc, nt 1628-1807) was the one that displayed a clear preference forhepatocytes with a moderate promoter strength.

It was assumed that the liver specificity of the expression was achievedprimarily by enhancer II. In order to produce a liver-specific promoterwith a higher activity, enhancer II was coupled with an enhancerlessminimum promoter. The TATA box and transcription start region (nt-55-+69) of the promoter of former cytomegalovirus transcripts were usedas a minimum promoter. The artificially produced promoter (EIImCMV)reaches more than 25% of the CMV promoter activity in hepatocytes, thusbeing classified as a strong promoter. The selectivity for hepatocytesis largely preserved. The promoter is also highly active the in primaryhepatocytes of mouse, pig and man. The long-term activity in hepatocytesis decisive to the promoter's usability in gene therapy vectors. Thepromoter was fuised with the β-galactosidase gene and cloned in theepisomal expression vector pREP8 (in vitro gene) to assess the activity.Stable hepatocyte lines (HuH7) were produced with the episome. Theβ-galactosidase expression was followed over the course of 3 months, andduring this period a deterioration in its expression level was notdetected.

In the following, the CP, EIImCMV and CMV promoters were coupled withthe cDNA of the human LDL receptor.

The receptor is primarily expressed in the liver. A defective gene forthe receptor serves as the basis for the hereditary disease "familyhypercholesterolaemia". The disease may be treated by the purposefultransfer of an active LDL receptor gene to the liver.

The expression unit consisting of the respective promoter and the LDLreceptor cDNA was inserted into the adenovirus transfer plasmid pdE1sp1Ain the two possible orientations. Recombinable adenoviruses wereproduced by cotransfection of the adenovirus genome (pJM17) into thehelper cell line HEK293. Viruses from individual recombination eventswere isolated by a plaque assay, reproduced in HEK293 cells, purifiedfrom cell lysate by a two-fold sedimentation in a CsCl₂ gradient and thetitre of the virus stocks was ultimately determined.

Testing of the promoter activity and the specificity after an adenoviralgene transfer in vitro

The liver cell lines HepG2 and HuH7 and the non-liver cell lines HeLaand CV1 were infected by 50 viruses/cell and the expression of the LDLreceptor gene was detected three days after the infection on RNA levelby RNAse protection and on protein level by Western Blot.

Whereas the activity of the gene controlled by the CMV promoter achieveda similar level in all examined cell lines a high expression of CP andEIImCMV promoters was only detected in hepatocyte cell lines (FIG. 3).The orientation of the expression unit in the virus has onlyinsignificant effects on the expression level. The activity of theHBV/CMV hybrid promoter achieved approximately 30 percent of theactivity of the CMV promoter.

Testing of the promoter activity and the specificity in vivo

To test the activity of the promoters in vivo, 2×10⁹ adenoviruses(Ad5-CMVLDLR, Ad5-E2mCVLDLR or the controlling vector Ad5-SVbg) werealways applied to the tail vein of mice (breeding race NMRI). Four daysafter infection the animals were killed, and the liver, lungs, diaphragmand kidney were extracted. The organs were powdered in liquid nitrogenand used for winning RNA and DNA. Southern Blot analysis has shown thatan essential part of the viruses infected the liver, while in the otherorgans only a few viral DNA were detected. However, part of the task wasto reliably detect insignificant promoter activities outside the liver.Thus, the lungs of further groups of mice were infected by intranasalapplication, and the viruses were injected into the muscles of a 3^(rd)group of animals. By using Southern Blotting techniques animals, whichwere detected to sustain comparable quantities of viral DNA in theinfected organ, were examined for transcripts of the human LDL receptorin a "RNAse protection assay". Whereas the expression of the CMVpromoter was detected in all three tissues, an expression of the EIImCMVpromoter could be only detected in the liver but not in the muscles andlungs. The quality and quantity of the isolated RNA were verified forthe ubiquitously active GAPDH gene by a "RNAse protection assay". As theCMV promoter showed only an insignificant activity in lungs and muscles,a highly sensitive assay was set up for competitive RT-PCR. Thereby, thesame quantities of RNA from the respective organ were mixed withincreasing quantities of a shortened LDL receptor RNA (competitor) whichwas synthesised in vitro, transcribed in reverse order and a LDLreceptor fragment was amplified in a PCR. When the quantity ofcompetitor RNA increases, the signal strength of the shortened PCRproduct is intensified, while an increased quantity of the longer PCRproduct corresponds to a decline in cellular RNA. Assuming theintensities of both bands are identical in the agarose gel, the quantityof the competitor provides information on the content of LDL receptorRNA in the tissue.

The results of competitive RT-PCR confirm those obtained by the "RNAseprotection assay". The activity of the EIImCMV promoter exceeds that ofthe CMV promoter in the liver. Yet, in muscles and lungs it lags behindthat of the CMV promoter by a factor of 10-30.

Thus, it is concluded that adenovirus vectors were constructed whichallow a high expression of the LDL receptor specifically in the liver,even if the virus is administered throughout the system. Moreover,negative effects of expression in cells outside the liver are excluded.

We claim:
 1. Liver-specific adenovirus expression vector comprising anadenovirus genome having inserted therein an expression unit, theexpression unit comprisinga therapeutic liver gene operatively coupledwith a liver-specific promoter comprising enhancer elements of thehepatitis B virus and an enhancerless minimum promoter activated by saidenhancer element.
 2. The vector according to claim 1 wherein theexpression unit is bordered in 3' and in 5' by scaffold attach regionelements.
 3. The vector according to claim 1, wherein said vectorcomprises a cDNA encoding a non-defective form of a gene associated witha disease condition.
 4. The Vector according to claim 1, whereinenhancer II of the hepatitis B virus is used as one of said enhancerelements.
 5. The vector according to claim 1, wherein positions1628-1807 on the genome of the hepatitis B virus of subtype ayw are usedas one of said enhancer elements.
 6. The vector according to claim 1,wherein a functional portion of the immediate early promoter of thehuman cytomegalovirus is used as the enhancerless minimum promoter. 7.The vector according to claim 1, wherein the expression unit is insertedin the El region of the adenovirus genome.
 8. The vector according toclaim 3, wherein the cDNA encodes the human LDL receptor is used as thetherapeutic gene.
 9. The vector according to claim 6, wherein thefunctional portion of said cytomegalovirus promoter comprises the TATAbox, the "cap site"and the 5' non-translated region.
 10. The vectoraccording to claim 7 wherein the adenovirus genome is selected from thegroup consisting of the adenovirus subtype 2 genome and adenovirussubtype 5 genome.